Members of the class Actinobacteria have been extensively studied because of their ability to produce pharmaceutically useful compounds. In order to construct a natural product library for bioactivity screening, we isolated a diverse variety of actinomycetes from marine resources, including marine sponges and tunicates, and mangrove soils, as well as the conventionally utilized terrestrial soils. As a result, we discovered novel compounds such as the anti-influenza compound JBIR-68,1 promothiocin derivatives JBIR-83 and -84,2 naphthablin analogs JBIR-79 and -80,3 and new angucyclines JBIR-90, -91, -92, -93 and -116.4 Further screening resulted in the isolation of two new cytotoxic compounds, JBIR-76 (1) and JBIR-77 (2), in addition to the antioxidative naphthoquinone-like polyketide, designated JBIR-85,5 from the culture of Streptomyces sp. RI-77 obtained from a soil sample collected in Okinawa Prefecture, Japan. This paper describes the fermentation, isolation, structural elucidation and brief biological activities of 1 and 2.

Streptomyces sp. RI-77 was isolated from a soil sample collected in Shuri, Okinawa Island, Japan, and cultivated in 50-ml test tubes containing 15 ml of seed medium composed of 1.0% starch (Kosokagaku, Tokyo, Japan), 1.0% Polypepton (Nihon Pharmaceutical, Tokyo, Japan), 1.0% molasses (Dai-Nippon Meiji Sugar, Tokyo, Japan) and 1.0% meat extract (Extract Ehlrich, Wako Pure Chemical Industry, Osaka, Japan pH 7.2, adjusted before sterilization). The test tubes were shaken on a reciprocal shaker at 27 °C for 2 days (320 r.p.m.). Aliquots (2.5 ml) of the culture were transferred into 500-ml baffled Erlenmeyer flasks filled with 100 ml of a production medium consisting of 0.9% oatmeal (Quaker, Chicago, IL, USA), 0.15% malt extract (Becton, Dickinson and Company; Franklin Lakes, NJ, USA), 0.09% yeast extract (Becton, Dickinson and Company), 0.9% soluble starch (Kosokagaku), 1.1% 3-(N-morpholino)propanesulfonic acid (Nacalai Tesque, Kyoto, Japan), 1.0% D-glucose (Kanto Chemical, Tokyo, Japan), 12 mg MgSO4, 30 mg NaCl and 150 mg CaCO3 at pH 7.0 (adjusted before sterilization), and cultured on a rotary shaker (180 r.p.m.) at 27 °C for 5 days.

After fermentation, the fermentation broth (2 l) was centrifuged, and the pelleted mycelial cake was extracted with acetone (800 ml) two times. The mycelium was removed by filtration, and the acetone extract was evaporated under reduced pressure. The residual aqueous concentrate was partitioned between EtOAc and water (200 ml × 3). The separated organic layer was dried over Na2SO4 and concentrated in vacuo. The residue (510 mg) was applied on a normal-phase medium-pressure liquid column (Purif-Pack SI-30; Shoko Scientific, Yokohama, Japan), and the column was successively eluted using a hexane-EtOAc solvent system (0, 5, 10, 20 and 25% EtOAc) followed by a CHCl3–MeOH solvent system (0, 2, 5, 10 and 20% MeOH). The 10% MeOH-eluted fraction (38.0 mg) was further purified by preparative RP-HPLC using a CAPCELL PAK C18 MGII column (5.0 μm, 20 i.d. × 150 mm; Shiseido, Tokyo, Japan) with a 2996 photodiode array detector (Waters, Milford, MA, USA) and a 3100 mass detector (Waters) developed with 55% aqueous MeOH (flow rate, 10 ml min−1) to yield JBIR-76 (1, 1.5 mg, retention time (Rt)=13.2 min) together with known compound JBIR-855 (0.4 mg, Rt=19.5 min). The 5% MeOH-eluted fraction (70.5 mg) was purified by preparative RP-HPLC using a CAPCELL PAK C18 MGII column with 68% aqueous MeOH (flow rate: 10 ml min−1) to yield JBIR-77 (2, 1.4 mg, Rt=16.3 min).

JBIR-76 (1) was isolated as a colorless oil: [α]25D +46.0, (c 0.1, MeOH); UV λmax nm (log ɛ): 327 (3.98) in MeOH; IR (attenuated total reflection) vmax cm−1: 3735, 1770 and 1650. The molecular formula of 1 as C15H14O7 was determined by positive high-resolution-electrospray ionization (HRESI)-MS (m/z 307.0833, calculated for C15H15O7, 307.0818). The structure of 1 was established on the basis of its double-quantum-filtered-COSY and constant-time heteronuclear multiple-bond correlation (CT-HMBC)6 spectra (Figure 1b). The 1H and 13C NMR spectroscopic data for 1 are shown in Table 1.

Figure 1
figure 1

(a) Structures of JBIR-85, JBIR-76 (1) and −77 (2). (b) Correlations in CT-HMBC (arrows) spectra of 1.

Full size image
Table 1 13C (150 MHz) and 1H (600 MHz) NMR spectroscopic data for JBIR-76 (1) in DMSO-d6 and 13C (125 MHz) and 1H (500 MHz) NMR spectroscopic data for JBIR-77 (2) in acetone-d6
Full size table

The 10 sp2 13C signals among 15, including two methoxy carbons, indicated that 1 consisted of a highly conjugated skeleton. The strong meta-couplings from an aromatic proton H-5 (δH 6.62) to aromatic carbons C-7 (δC 135.0) and C-8a (δC 110.6), in addition to 1H-13C long-range couplings to C-4a (δC 138.7) and C-6 (δC 158.3) observed in the CT-HMBC spectrum established a benzene ring moiety. The 1H-13C long-range coupling observed between a methoxy proton 7-OMe (δH 3.71) and a quaternary carbon C-7 revealed that this methoxy group was substituted at the position of C-7. The 1H-13C long-range couplings from a phenolic hydroxy proton 8-OH (δH 13.20), which was considered to be hydrogen-bonded with a carbonyl carbon at the peri position, to aromatic carbons C-7, C-8 (δC 158.3) and C-8a established the penta-substituted benzene ring moiety. By taking into consideration its 13C chemical shift at C-6 (δC 158.3), an oxygen atom was determined to be substituted at C-6. Further, the 1H-13C long-range couplings from a singlet hydroxymethyl proton H2-10 (δH 4.78, δC 55.3) to aromatic carbons C-1 (δC 160.0) and C-9a (δC 116.5), and from an aromatic proton H-3 (δH 8.02) to C-1, C-3a (δC 122.1) and C-9a established a 2,3,4-trisubstituted furan moiety, and that a hydroxymethyl residue was substituted at the position of C-1. The 1H-13C long-range couplings from an oxymethine proton H-4 (δH 5.47) to C-3 (δC 141.2), C-3a, C-4a, C-5 (δC 111.1), C-8a and C-9a established a 6-6-5 ring system, as shown in Figure 1. The 1H-13C long-range coupling between a methoxy proton 4-OMe (δH 3.09) and C-4 (δC 68.6) revealed that the methoxy functional residue is substituted at the position of C-4. Finally, hydroxy groups at C-6 and C-10 were determined based on the molecular formula (Figure 1a).

JBIR-77 (2) was isolated as a colorless oil: [α]25D +27.8, c 0.1, MeOH; UV λmax nm (log ɛ): 324 (3.98) in MeOH; IR (attenuated total reflection) vmax cm−1 3520, 1750 and 1650. The molecular formula of 2 as C15H14O6 was determined by positive HRESI-MS (m/z 291.0863, calculated for C15H15O6, 291.0869). The IR and UV spectra of 2 were similar to those of 1. Most of the NMR spectroscopic data for 2 were also similar to those of 1 (Table 1). In the 1H NMR spectrum of 2, the singlet signal due to the oxymethylene group at H2-10 in 1 was replaced with a singlet methyl proton (δH 2.67). In addition, the oxymethylene carbon in 1 was replaced with a methyl carbon (δC 13.9 for C-10) in 2. These collective spectroscopic data proved 2 to be 10-dehydroxy 1 (Figure 1a).

The cytotoxic activities of 1 and 2 against human ovarian adenocarcinoma SKOV-3, malignant pleural mesothelioma Meso-1 and Jurkat cells were examined using a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) colorimetric assay (Cell Counting Kit-8; Dojindo, Kumamoto, Japan). All cell lines were seeded in 384-well plates at a density of 1000 cells per 20 μl per well and incubated at 37 °C in a humidified incubator with 5% CO2. After 72 h of treatment, 1 exhibited cytotoxic activities against SKOV-3, Meso-1 and Jurkat cells with IC50 values of 18.7, 11.3 and 7.6 μM, respectively. Compound 2 exhibited almost the same cytotoxic activities against SKOV-3, Meso-1 and Jurkat cells with IC50 values of 9.7, 33.8 and 3.6 μM, respectively.

In conclusion, we isolated two new modified naphthoquinones, JBIR-76 (1) and JBIR-77 (2), from the culture of Streptomyces sp. RI-77 obtained from a soil sample collected in Okinawa Prefecture, Japan. During the same period, the antioxidative naphthoquinone-like polyketide, designated JBIR-85,5 was also isolated. The structures of 1 and 2 consisted of a new skeleton, which is structurally related to 5,8-dihydroxy-1-hydroxymethylnaphtho[2,3-c]furan-4.9-dione isolated from the roots of Bulbine capitata.7 Biosynthetic studies of these compounds are now underway.